The EMBO Journal vol.6 no.3 pp.549-554, 1987

In vitro expression of a full-length DNA copy of cowpea mosaicvirus B RNA: identification of the B RNA encoded 24-kd proteinas a viral protease

Jan Verver, Rob Goldbachl, Juan A.Garcia2 and PieterVos

Department of Molecular Biology, Agricultural University, De Dreijen 11,6703 BC Wageningen, 'Department of Virology, Agricultural University,Binnenhaven 11, 6709 PD Wageningen, The Netherlands, and 2Centro deBiologia Molecular, Universidad Autonoma de Madrid, 28049 Madrid,Spain

Communicated by A.-L.Haenni

Double-stranded cDNA was synthesized from B componentRN9A ofcowpea mosaic virus and cloned into appropriate vec-tors. Using four clones, together representing the entire BRNA sequence, a full-length DNA copy was constructed andsubsequently positioned downstream of a phage SP6 or T7promoter. RNA molecules transcribed from this full-size DNAcopy using SP6 or T7 RNA polymerase were efficiently trans-lated in rabbit reticulocyte lysates into a 200-kd polypeptidesimilar to RNA isolated from viral B components. Moreoverthis polypeptide was rapidly cleaved into 32-kd and 170-kdpolypeptides, exactly like the 200-kd polypeptide encoded byviral B RNA. In vitro transcription and translation of a DNAcopy in which an 87-bp-long deletion in the coding sequencefor the 24-kd polypeptide was introduced revealed that the24-kd polypeptide bears the proteolytic activity involved inthe primary cleavage of the B RNA-encoded polyprotein.Key words: cowpea mosaic virus/cDNA/in vitro transcription/invitro translation/viral protease

IntroductionCowpea mosaic virus (CPMV) is a plant virus with a genomeconsisting of two separately encapsidated, positive-sense RNAmolecules designated M and B RNA (for a recent review seeGoldbach and Van Kammen, 1985). The viral RNAs are charac-terized by a small protein, denoted VPg, covalently linked totheir 5'-end (Stanley et al., 1978; Daubert et al., 1978) and apoly(A)-tail at their 3'-end (El Manna and Bruening, 1973). Theyare both translated into large polypeptides ('polyproteins') fromwhich the functional proteins are derived by proteolytic cleavages(Rezelman et al., 1980; Goldbach et al., 1981). In all theserespects CPMV resembles the animal picomaviruses. The markedsimilarity in genome organization and the sequence homologyamong their non-structural proteins, moreover, suggest a geneticrelationship (Franssen et al., 1984a).B RNA is translated both in vivo and in vitro into a 200-kd

polyprotein, which is rapidly cleaved into a 170-kd and a 32-kdpolypeptide. The 170-kd polypeptide is then further processedinto polypeptides with sizes of 110, 87, 84, 60, 58, 24 and 4 kd(VPg) (Rezelman et al., 1980; Goldbach and Rezelman, 1983;Franssen et al., 1984b and Figure 1). These cleavage productshave been mapped on the 200-kd polyprotein by sequencing oftheir amino-terminal ends (Wellink et al., 1986: Zabel et al.,1984). The results revealed that three different types of cleavagesites are employed during the processing of the B RNA-encoded

polyprotein: glutamine-methionine (lx), glutamine-serine (2x)and glutamine -glycine (lx) dipeptide sequences.

Inhibition studies using antisera directed against various viralproteins strongly suggested that the 32-kd protein (see Figure1) represents a protease involved in the processing of the uniqueglutamine -methionine cleavage site in the M RNA-encodedpolyproteins (Franssen et al., 1984c). Hence involvement of thisprotease in cleavage at the glutamine -methionine site of the Bpolyprotein is also very probable. Time course translations haveprovided indications that the 24-kd polypeptide encoded by a BRNA represents a second viral protease (Franssen et al., 1984b).This suggestion was further supported by the sequence homologyof this polypeptide to the picornaviral protease P-3C (Franssenet al., 1984a). We have now constructed and cloned a full-lengthDNA copy ofB RNA in order to introduce mutations on specificsites in this copy and to express mutagenized copies in vitro ac-cording to the approach described previously for M RNA (Voset al., 1984). Following this approach we have been able todemonstrate that the 24-kd protein encoded by B RNA indeedrepresents a protease involved in the primary cleavage of the200-kd polyprotein.

ResultsConstruction and molecular cloning of a full-length DNA copyof B RNADouble-stranded cDNA was synthesized from B RNA (VanWezenbeek et al., 1983) using an oligonucleotide homologousto the first 16 nucleotides of B RNA to prime second-strand syn-thesis. The DNA obtained was digested with suitable restrictionenzymes at positions which could be deduced from the nucleotidesequence of B RNA (Lomonossoff and Shanks, 1983). In this

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Fig. 1. Model for the translation of the B RNA of CPMV. The double-linedbar in the RNA refers to the long open reading frame running from startcodon at position 207 to a stop codon at position 5805 (Lomonossoff andShanks, 1983). VPg is indicated as a black square, other protein sequencesas single lines. Cleavage sites are indicated by open circles(glutamine-methionine), open triangles (glutamine-glycine) or closedtriangles (glutamine-serine) (Wellink et al., 1986).

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Fig. 2. Construction of a full-length DNA copy of CPMV B RNA. Panel A: cloning strategy for construction of a full-size DNA copy from four smallercDNA clones. The upper line represents the complete B RNA sequence with the double-lined bar indicating the long open reading frame. The positions ofrelevant recognition sites of BamHI (B), EcoRI (E), HindHI (H), Pstl (P), Sall (S), Sacl (Sac) and SniaI (Sma) are shown. In the various cDNA clonesobtained vector-derived sequences are indicated by a single line, B RNA-derived sequences by open boxes and the poly d(A-T) track of 55 residues as adashed box. Double-stranded cDNA was synthesized using oligo(dT) as primer for first strand synthesis and an oligonucleotide homologous to the first 16nucleotides of B RNA as primer for second strand synthesis. Digestion of the double-stranded cDNA with BamHI and Sacl and subsequent ligation into pUC8linearized with SmiaI and BamHI and pUC12 digested with Sacl and BamHI resulted in cDNA clones pCB93 and pCB22. Clone pCB93 contained a B RNA-specific insert from the BamHI site at position 3857 to the 3'-end including a poly d(A-T) track, cDNA clone pCB22 contained a viral cDNA sequence fromthe Sacl recognition sequence at position 2301 to the BamHI site at position 3857. In addition, double-stranded cDNA was digested with Sacl and EcoRI andligated into pUC12 linearized with Sacl and EcoRI. One of the cDNA clones obtained, pCB71, contained the EcoRI-SacI cDNA fragment, corresponding topositions 264-2301 of the B RNA sequence. To obtain a cDNA clone containing the ultimate 5'-end of the B RNA sequence, first strand synthesis wasprimed with a 388-bp KpnI fragment (positions 2746-3034), derived from cDNA clone pCB22. Double-stranded cDNA synthesized in this way was digestedwith EcoRI and subsequently ligated into EcoRI- and SmiaI-digested pUC8 resulting in clone pCB5, which contained a cDNA insert corresponding to positions1-264 of the B RNA sequence. cDNA clones pCB5, pCB71, pCB22 and pCB93, together representing the entire B RNA-sequence, were used to construct afull-size DNA copy. For this purpose, first a cDNA clone containing the first 2301 nucleotides of B RNA was constructed (pCB814) by joining the cDNAinserts of clone pCB5, obtained upon digestion with Sall and EcoRI, and clone pCB71, obtained upon digestion with EcoRI and Sacl, via a three-pointligation into pUC12 linearized with Sail and Sacl. A unique Sall site was introduced in clone pCB93 downstream of the poly d(A-T) track. Firstly, the Sallsite already present in this construct was removed by digesting the DNA with Sall and subsequent religation after filling-in the recessed ends by the Klenowfragment of E. coli DNA polymerase I. Secondly, the resulting clone pCB933 was partially digested with EcoRl, made blunt-end with SI nuclease and thelinearized DNA, obtained by digestion of one of the two EcoRI sites eluted from the gel (Tautz and Renz, 1984). Finally Sall linkers were coupled to theDNA, cohesive ends were generated by digestion with Sall and the DNA was religated. One of the clones obtained in this way, pCB9331, contained a uniqueSall site downstream of the poly d(A-T) track as intended. The BamHI-Sall fragment from this clone and the SacI-BamHI fragment from clone pCB22were joined via a three-point ligation with Sacl and Sall digested pSP65 to produce clone pSPB119, containing a cDNA copy corresponding to the 3'-end ofB RNA from position 2301 downstream. Finally a full-size cDNA clone was constructed, denoted pSPB20, by joining of the BanHI-SacI fragment ofpCB814 with the SacI-PstI fragment of pSPBl19 in a three-point ligation with BamHI- and PstI-digested pSP65. pSPB20 has a total length of 8.95 kb andthe B RNA-specific cDNA can be excised from this plasmid with SmaI and Sall (see also panel B). This 5.95-kb SmaI-Sall fragment, containing the full-length DNA copy, was inserted into plasmid pT7-I digested with SmaI and Sall, resulting in clone pTB3 (not shown). Using this clone B RNA-liketranscripts could be synthesized initiated at the T7 promoter present in the construct. Panel B: schematic representation of the full-length DNA copy. Thelong open reading frame with the positions of various B RNA-specific proteins is indicated by a large open bar, other B RNA-specific sequences are indicatedby double lines. The region in the DNA copy used in the construction of a mutant with a deletion in the coding region for the 24-kd polypeptide is presentedenlarged (see Materials and methods). Relevant recognition sites for restriction enzymes are indicated: Hae (HaeII), K (KpnI), Sau (Sau3A), RV (EcoRV),for other enzymes see panel A.

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Fig. 3. Native 1.2% agrarose gel with reaction products of several in vitrotranslation assays using T7 RNA polymerase. On the right-hand side thepositions of template DNA and RNA transcripts are shown. On the left-hand side the positions of M and B RNA isolated from virus particles areindicated. Lane 1 shows M and B RNA isolated from virus particles. Lanes2 and 5 show linearized template DNA of pTB3 and pTB3A I respectively.Reaction products of in vitro transcriptions with pTB3 and pTB3A 1 areshown without DNase treatment (lanes 3 and 6 respectively) or DNasetreated (lanes 4 and 7 respectively). Reaction conditions were as describedin Materials and methods using 20 units T7 RNA polymerase per /kg oftemplate DNA. Template DNA was removed by DNase treatment asdescribed previously (Vos et al., 1984).

way a set of restriction fragments was generated from the cDNAsynthesized which, after cloning in suitable vectors, representedthe entire B RNA sequence. After verifying their integrity byrestriction enzyme and/or sequence analysis the cDNA insertsof four different clones were selected and ligated to generate afull-length cDNA clone of B RNA. The strategy of this multi-step cloning procedure is outlined in Figure 2A.The resulting clone pSPB20 had a total length of 8.95 kb with

a unique SalI-site downstream of a poly d(A-T) track of 55 bpthat could be utilized to synthesize run-off transcripts initiatedat the SP6-promoter by phage SP6 RNA polymerase (Green etal., 1983; Vos et al., 1984). Alternatively the viral insert of clonepSPB20 was positioned downstream of a phage T7 promoter,resulting in clone pTB3, to allow in vitro transcription by RNApolymerase from phage T7 instead of SP6 (Figure 2).In vitro expressionTo generate run-off transcripts plasmids pSPB20 and pTB3 werelinearized downstream of their poly d (A-T) track by digestionwith Sail (Figure 2) and trancribed using SP6 or T7 RNApolymerase, repectively (Vos et al., 1984; Davanloo et al., 1984)(see Figure 3). Discrete transcripts of 6.0 kb were produced con-taining, in addition to B RNA-specific sequences, - 35 extranucleotides to the 5' end, derived from the SP6 or T7 promoterand multiple cloning site, and nine nucleotides at the 3' end ofthe 55 residue-long poly(A) tail. A further difference with B RNAfrom virions was the absence at the 5 '-end of the genome-linkedprotein VPg. Depending on the amount of SP6 or T7 polymeraseadded the efficiency of the in vitro transcription reactions couldbe increased, yielding up to 20 yg ofRNA from 1 itg of templateDNA. Occasionally transcripts were capped by addition of thecap precursor m7GpppG to the transcription reaction (Konarskaet al., 1984). The efficiency of capping depended on the ratioof m7GpppG to GTP in the reaction mixture. In the case of SP6

Fig. 4. In vitro translation products of transcripts obtained from variousCPMV-specific cDNA clones using SP6 or T7 polymerase. Numbers at theleft-hand side indicate the sizes (in kd) of marker proteins used: myosin,210; phosphorylase b, 100 and 92.5; bovine serum albumin, 68; ovalbumin46; carbonic anhydrase, 30. Numbers at the right-hand side correspond tothe positions of relevant viral proteins encoded by natural M or B RNA.Panel A: in vitro translation products of viral B RNA and of transcriptssynthesized by SP6 RNA polymerase from pSPB20 or by T7 RNApolymerase from pTB3, with (+ cap) or without a cap structure at the 5'end. Panel B: in vitro translation products of viral B RNA and oftranscripts synthesized by SP6 or T7 RNA polymerase using 'wild type'DNA copies as template DNA (pSPB20, pTB3) or DNA copies in which adeletion in the coding region for the 24-kd polypeptide was introduced(pSPB20A1 and pTB3A1). Panel C: in vitro translation products of viral MRNA, untreated (control) or incubated with unlabelled translation productsof viral B RNA, or of transcripts from pSPB20 or pSB20 1. M RNA wastranslated in the presence of [3 S]methionine as described previously(Goldbach et al., 1981) and after 1 h a 3 times excess of unlabelledtranslation products of viral B RNA or B RNA transcript was added andincubation continued at 30°C for another 5 h. In the case of the controlsample, incubation was also continued for 5 h. The lane between panels Band C shows the [35S]methionine-labelled proteins extracted from CPMV-infected protoplasts.

polymerase capping was very efficient giving 50% capped trans-cripts with equal amounts of GTP and m7GpppG. For the T7polymerase a four times molar excess of m7GpppG to GTP hadto be used to achieve 50% capping (results not shown).The in vitro transcripts obtained were translated in rabbit

reticulocyte lysates resulting in efficient production of a 200-kdprotein, co-migrating with the 200-kd polyprotein produced fromnatural B RNA (Figure 4A). Moreover this 200-kd polypeptidewas cleaved into two polypeptides with sizes of 170 and 32 kd,similar to the primary cleavage of B RNA-encoded 200-kdpolyprotein (Figure 4A). These results show that the DNA copycontained a single long open reading frame similar to viral BRNA and that the translation product contains the proteolytic ac-tivity for cleavage of the polyprotein at the right position. Nodifferences were observed between the in vitro translation pro-ducts of in vitro RNAs synthesized by T7 or SP6 RNApolymerase. The presence of the cap structure at the 5' end of

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the in vitro transcripts did not significantly increase the transla-tional activity of the transcripts, which is in contrast to resultsobtained with several in vitro synthesized eukaryotic mRNAs(Krieg and Melton, 1984). The minor differences in the amountsof translation products obtained (Figure 4A) are probably dueto variations in RNA concentration in the in vitro translation reac-tions. The relatively large amounts of smaller translation pro-ducts obtained with viral B RNA probably result from translationof degraded RNA molecules.Assignment ofproteolytic activity to the 24-kd protein encodedby B RNAThe availability of a full-length DNA copy of B RNA enabledus to study the genetic organization of B RNA by in vitro ex-pression ofDNA copies, modified by site-directed mutagenesis.One of the features of CPMV expression that lends itself wellfor in vitro analysis is the proteolytic processing of the primarytranslation products. This process occurs very reliably upon invitro translation in rabbit reticulocyte lysates (Pelham, 1979;Franssen et al., 1984b,c; Peng and Shih, 1984). The 24-kdpolypeptide encoded by B RNA may have an important role inthe proteolytic processing reactions since this protein shows se-quence homology to the picornaviral protease P-3c (Franssen etal., 1984a).To test whether the 24-kd polypeptide indeed possesses proteo-

lytic activity we introduced a small deletion in the coding regionfor this protein within the DNA copy of B RNA and determinedpossible defects in proteolytic cleavage of the translation productof this deletion mutant. For this purpose an 87-bp Sau3A frag-ment, corresponding to positions 3240-3327 of the B RNA se-quence, was excised from the full-size DNA copy (Figure 2Band Materials and methods). This deletion did not change thereading frame, but resulted in the loss of the coding sequencefor 29 amino acids of the 24-kd polypeptide. The resulting dele-tion mutant, denoted pSPB20OA1 or pTB3AI depending on thechoice of the promoter, was subjected to subsequent in vitrotranscription and translation after linearization with Sal. Com-pared to the primary translation products of both viral B RNAand transcripts of clones pSPB20 or pTB3, the translation pro-duct of the deletion mutant was, as expected, slightly smaller(see Figure 4B). Furthermore this product did not undergo thefaithful proteolytic processing as observed with the translationproducts of pSPB20 and pTB3 (figure 4B). This finding indicatesthat the 24-kd polypeptide indeed represents a proteolytic enzyme,involved in the primary cleavage of the 200-kd viral polypro-tein, which occurs at a glutamine -serine pair (Wellink et al.,1986).The 32-kd protein released by the primary cleavage has been

reported to represent also a viral protease, involved in the cleav-age of the M RNA encoded polyproteins at a glutamine -meth-ionine site (Franssen et al., 1982, 1984c). We were interestedin the effect of the deletion in the 24-kd protease, i.e. blockingof the release of the 32-kd protease, on the activity of this secondprotease. For this purpose in vitro translation products of viralB RNA or of transcripts from the cDNA clones pSPB20 andpSPB20A1, respectively, were mixed with the M RNA-encodedpolyproteins and incubated for 5 h at 30°C (Figure 4C). Signifi-cant processing of the M RNA-encoded 95-kd polyprotein topolypeptides of 60 and 48 kd (Franssen et al., 1982) was observedwhen in vitro translation products of viral B RNA or of transcriptfrom clone pSPB20 were added, but upon addition of transla-tion product from the mutant clone pSPB20 1 processing couldnot be detected (Figure 4C). A possible explanation for this effect

of the B RNA-encoded 24-kd polypeptide on the processing ofthe primary translation products of M RNA at a glutamine-methionine site is discussed below.

DiscussionVarious animal and plant RNA viruses use proteolytic process-ing to synthesize their proteins. Most of the current knowledgeon viral protein cleavage reactions is derived from studies withpicomaviruses (for recent review see Toyoda et al., 1986; Nicklinet al., 1986; Jackson, 1986) and CPMV (Goldbach and VanKammen, 1985). Until recently it was generally believed thatin the proteolytic processing of the picornaviral polyproteins, inaddition to the viral protease 3C, a cellular enzyme was involved(Korant et al., 1980; Burroughs et al., 1984). By analogy to thepicornaviruses a similar involvement of a host-encoded proteasein the cleavage of the CPMV polyproteins may be surmized. Inthis paper we report the construction of a full-length cDNA cloneof CPMV B RNA and its subsequent exploitation for studyingthe proteolytic processing of the CPMV translation products. Thein vitro transcription and translation experiments described heredemonstrate that the B RNA-encoded 24-kd polypeptiderepresents a viral protease, being involved in the primary cleavageof the 200-kd polyprotein, thereby excluding the possible involve-ment of a cellular enzyme in this process.

Bacteriophage SP6 RNA polymerase has been demonstratedto be very suitable for in vitro transcription of DNA templates(e.g. Green et al., 1983; Melton et al., 1984; Vos et al., 1984;Tabler and Sanger, 1985). In addition to SP6 RNA polymerasewe now also have used T7 RNA polymerase to synthesize RNAfrom templates provided with a T7 promoter fragment. RNA syn-thesis by T7 RNA polymerase also appeared to be very efficient,giving yields comparable with those of SP6 polymerase-dependentreactions and, furthermore, efficient capping of the RNAtranscripts could be achieved similar to the SP6 polymerase(Konarska et al., 1984). The advantage of the use of T7 insteadof SP6 RNA polymerase in in vitro transcription assays may bethe thorough understanding of the genetics of phage T7.Transcription of T7 genes under the control of T7 promoters hasbeen extensively studied (see for instance Dunn and Studier,1983). Moreover, plasmids are available containing the T7 RNApolymerase gene under the control of the XPL promoter, permit-ting simple, large-scale purification of the enzyme from induc-ed Escherichia coli cells (Tabor and Richardson, 1985; thispaper).

Recently the proteolytic cleavage sites in the 200-kd polypro-tein from B RNA have been identified (Wellink et al., 1986).Three types of cleavage sites are used for processing, i.e. glut-amine-serine, glutamine-glycine and glutamine-methioninedipeptide sequences. For processing of the M RNA-encoded poly-proteins only two of these three different cleavage sites are used:a glutamine -methionine dipeptide sequence to release the 60-kdcapsid protein precursor, and a glutamine-glycine dipeptidesequence to release the two capsid proteins from this precursor(Van Wezenbeek et al., 1983; Vos et al., 1984). Inhibition studiesusing antisera raised against various viral proteins and in vitroprocessing studies using partially purified proteins from Bcomponent-inoculated protoplasts strongly suggested that the32-kd protein encoded by B RNA (see Figure 1) is involved inthe cleavage of the M RNA-encoded polyproteins at the glut-amine-methionine site (Franssen et al., 1984b,c). If this pro-tein indeed represents a protease recognizing this site then it ismost probably also involved in cleavage of the single glutamine-

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methionine site in the B polyprotein, which is situated at the leftborder of the 24-kd polypeptide sequence (Figure 1).

Since we have now demonstrated that the 24-kd polypeptidealso possesses proteolytic activity, responsible for the primarycleavage of the 200-kd polyprotein at a glutamine -serine dipep-tide sequence, the question arises which of the two proteasesrecognizes the glutamine - glycine cleavage sites. Comparisonof the three types of cleavage sites used in the processing of theCPMV polyproteins shows that they fall into two categories.Although all cleavage sites share a glutamine residue at the firstposition of the dipeptide sequence the nature of the residue atthe second position is quite variable, being either small and polar(glycine and serine) or large and non-polar (methionine). It istherefore tempting to assume that the 32-kd polypeptiderecognizes only glutamine - methionine cleavage sites, while the24-kd polypeptide may be involved in cleavage of both theglutamine-serine and glutamine-glycine cleavage sites. Indeed,preliminary experiments seem to indicate that the 24-kd polypep-tide is also involved in the cleavage at a glutamine -glycine sitein the M RNA-encoded polyproteins (data not shown).

Surprizingly the small deletion in the coding region of the 24-kdpolypeptide also affected the processing of the M RNA-encodedpolyproteins at a glutamine -methionine site, which is supposedto be recognized by the 32-kd protease (Franssen et al., 1984c).While such processing was observed upon addition of transla-tion products from clone pSPB20 this was clearly not the casewith translation products from clone pSPB20A 1. The most plaus-ible explanation for this inhibitory effect is that release of the32-kd polypeptide from the 200-kd polyprotein (by the 24-kd pro-tease) is a prerequisite for the 32-kd polypeptide to become alsoproteolytically active. Clearly further experiments are necessaryto confirm this latter hypothesis and to understand fully how thetwo different B RNA-encoded proteases co-operate in the releaseof all mature proteins from the viral polyproteins.

Materials and methodsNucleic acids and enzymesCPMV was propagated in Vigna unguiculata L., 'California Blackeye' and Bcomponents were purified as described previously (Klootwijk et al., 1977). CPMVB RNA was extracted from purified B components as described by Zimmern(1975) or Davies et al. (1978). Plasmid DNA isolations on large or small scalewere performed by the alkaline lysis method (Birnboim and Doly, 1979). Allenzymes used were purchased from Boehringer Mannheim B.V. or AnglianBiotechnology Corp. and were used according to the manufacturer's indications.T7 RNA polymerase was isolated as described below. Plasmid pT7-1 was pur-chased from United States Biochemical Corp. The 16-mer d(TATTAAAAT-CAATACA) was synthesized by the chemical phosphotriester method (Van derMarel et al., 1982).Molecular cloningDouble-stranded cDNA was synthesized from CPMV RNA as described previously(Van Wezenbeek et al., 1983). DNA fragments were joined by T4 DNA ligaseand transformed into competent E. coli HBIOI cells as described by VanWezenbeek et al. (1983). DNA fragments used for ligation were generally purifiedeither by extraction from agarose gels (Tautz and Renz, 1984; Weislander, 1979)or by elution from polyacrylamide gel slices (Maxam and Gilbert, 1977). Recom-binant DNA clones were analysed by restriction enzyme mapping and/or nucleotidesequencing (Sanger et al., 1977, 1980).Introduction of a deletion in the full-length DNA copy of B RNA

A Haefll-EcoRI fragment (corresponding to positions 2815 -3865 of the B RNAsequence) (see Figure 2B) was isolated from a full-length cDNA clone of B RNA(pSPB20) and inserted in plasmid pUC9 previously digested with SnaI and EcoRI.The resulting clone, pLB281, was cut with KpnI and EcoRV, both having uniquerecognition sites in this plasmid, yielding a small DNA fragment of 301 bp corre-

sponding to positions 3134 (KpnI) to 3435 (EcoRV) of the B RNA sequence(see Figure 2B), together with a large fragment. The 301-bp fragment was subse-quently digested with Sau3A resulting in three fragments of 106 bp (KpnI, 3134,

to Sau3A, 3240). 87 bp (Sau3A, 3240, to Sau3A, 3326) and 108 bp (Sau3A,3327, to EcoRV, 3435). In a three-point ligation the 106-bp KpnI-Sau3A frag-ment and 108-bp Sau3A-EcoRV fragment were joined together to the largeKpnI-EcoRV fragment of pLB281. In this way a clone was constructed,pLB28/ASau87, that contained a HaeIlI-EcoRI fragment (corresponding to posi-tions 2815 -3865 of the B RNA sequence), missing the 87-bp Sau3A fragment(positions 3240-3327). To construct a full-length cDNA clone of B RNA con-taining this deletion, the KpnI-EcoRI fragment (positions 3134-3865) of clonepLB281ASau87 was exchanged for the corresponding fragment of pSPB20,yielding clone pSPB20A1. The SmiaI-Sall fragment of pSPB20A1, containingthe full-size DNA copy of B RNA with the desired deletion was also positioneddownstream of the T7 promoter in plasmid pT7-1 in a similar way as decribedfor clone pTB3 (see Results and Figure 2) yielding clone pTB3A1.Purification of 77 RNA polymeraseAs source for the purification of T7 RNA polymerase plasmid pT7P2 was usedin E. coli strain NFl (Bernard et al., 1979). Isolation was according to Taborand Richardson (1985) with some modifications. Plasmid pT7P2 contained aBamHI-EcoRI fragment from the low copy number plasmid pGPl (a kind giftof Stanley Tabor; described in Tabor and Richardson, 1985) cloned into the highcopy number plasmid pUC8. This fragment contained the T7 RNA polymerasegene under control of the XPL promoter. Due to a temperature-sensitive cIrepressor gene in bacterial strain NFl (Bernard et al., 1979) synthesis of T7polymerase can be triggered by raising the temperature from 28 to 42°C.

Cells were grown at 28°C until an OD6M of 0.4 was reached and then in-cubated for 90 min at a temperature of42°C. Cells were collected by centrifuga-tion and lysed according to the procedure of Butler and Chamberlin (1982). Thelysate was centrifuged for 30 min at 20 000 g and (NH4)2SO4 was added to thesupernatant to a saturation level of 30%. The (NH4)2SO4 suspension was cen-

trifuged for 30 min at 20 000 g and the pellet fraction containing most of theT7 RNA polymerase was further purified as described by Tabor and Richardson(1985).In vitro transcription and translationIn vitro transcription using SP6 polymerase was performed as described previously(Vos et al., 1984). In vitro transcription with T7 RNA polymerase was carriedout in 100 /Al containing 40 mM Tris-HCI pH 8.0, 20 mM MgCl2, 10 mMDTT, 100 td/ml BSA, 1 mM of each four NTPs, 5 Ag linearized template DNA,5-50 U of T7 polymerase and 50 U of RNasin. To synthesize capped transcriptsthe GTP concentration was lowered to 0.2 mM and the cap precursor m7GpppGwas added to a final concentration of 2 mM.

Aliquots of the transcription mixture were translated directly, without anypurification, in rabbit reticulocyte lysates using [35S]methionine as radioactiveamino acid as described previously (Goldbach et al., 1981). In vitro translationproducts were analysed on a 12.5% SDS-polyacrylamide gel (Franssen et al.,1982).

AcknowledgementsWe thank Jacques van Boom for synthesis of the oligodeoxynucleotide, RichardJackson for the gift of the rabbit reficulocyte lysates, Ab van Kammen for criticalreading of the text, Piet Madern for artwork and photography and Gre Heitkonigfor typing the manuscript. This work was supported by The Netherlands Foun-dation for Chemical Research (SON) with financial aid from The NetherlandsOrganization for the Advancement of Pure Research (ZWO).ReferencesBernard,H.U., Remaut,E., Vickers Hersfield,M.V., Das,H.K., Helinski,D.R.,

Yanofsky,C. and Franklin,N. (1979) Gene, 5, 59-76.Birnboim,H.C. and Doly,J. (1979) Nucleic Acids Res., 7, 1513-1523.Burroughs,J.N., Sanger,D.V., Clarke,B.E., Rowlands,D.J., Billian,A. and Col-

len,D. (1984) J. Virol., 50, 878-883.Butler,E.T. and Chamberlin,M.J. (1982) J. Biol. Chem., 257, 5772-5778.Daubert,S.D., Bruening,G. and Najarian,R.C. (1978) Eur. J. Biochem., 92,45-51.

Davanloo,P., Rosenberg,A.H., Dunn,J.J. and Studier,F.W. (1984) Proc. Natl.Acad. Sci. USA, 81, 2035-2039.

Davies,J.W., Verver,J.W.G., Goldbach,R.W. and Van Kammen,A. (1978)Nucleic Acids Res., 5, 4643 -4661.

Dunn,J.J. and Studier,F.W. (1983) J. Mol. Biol., 166, 477-535.El Manna,M.M. and Bruening,G. (1973) Virology, 56, 198-206.Franssen,H., Goldbach,R., Broekhuijsen,M., Moerman,M. and Van Kammen,A.

(1982) J. Virol., 41, 8-17.Franssen,H., Leunissen,J., Goldbach,R., Lomonossoff,G. and Zimmern,D.

(1984a) EMBO J., 3, 855-861.Franssen,H., Goldbach,R. and Van Kammen,A. (1984b) Virus Res., 1, 39-49.Franssen,H., Moerman,M., Rezelman,G. and Goldbach,R. (1984c) J. Virol.,

50, 183-190.

553

J.Verver et al.

Goldbach,R. and Rezelman,G. (1983) J. Virol., 46, 614-619.Goldbach,R.W. and Van Kammen,A. (1985) In Davies,J.W. (ed.) Molecular

Plant Virology. CRC Press, Boca Raton, FL, Vol. II, pp. 83-120.Goldbach,R.W., Schilthuis,J.G. and Rezelman,G. (1981) Biochem. Biophys. Res.

Commun., 99, 89-94.Green,M.R., Maniatis,T. and Melton,D.A. (1983) Cell, 432, 681-694.Jackson,R.J. (1986) Virology, 149, 114-127.Klootwijk,J., Klein,I., Zabel,P. and Van Kammen,A. (1977) Cell, 11, 73-82.Konarska,M.M., Padgett,R.A. and Sharp,P.A. (1984) Cell,, 38, 731-736.Korant,B.D., Chow,N.L., Lively,M.O. and Powers,J.C. (1980) Ann. NYAcad.

Sci., 343, 304-318.Krieg,P.A. and Melton,D.A. (1984) Nucleic Acids Res., 11, 7057-7070.Lomonossoff,G.P. and Shanks,M. (1983) EMBO J., 2, 2253-2258.Van der Marel,C.A., Wille,G., Marugg,J.E., De Vroom,E., Tromp,M., Van

Boekel,C.A.A. and Van Boom,J.H. (1982) Rec. Trav. Chim. Pays-Bas, 10,239-241.

Maxam,A.M. and Gilbert,W. (1977) Proc. Natl. Acad. Sci. USA, 74, 560-564.Melton,D.A., Krieg,P.A., Rebagliati,M.R., Maniatis,T., Zinn,K. and

Green,M.R. (1984) Nucleic Acids Res., 12, 7035 -7056.Nicklin,M.J.H., Toyoda,H., Murray,M.G. and Wimmer,E. (1986) Biotechnology,

4, 33-42.Pelham,H.R.B. (1979) Virology, 96, 463-477.Peng,X.X. and Shih,D.S. (1984) J. Biol. Chem., 259, 3197-3201.Rezelman,G., Goldbach,R. and Van Kammen,A. (1980) J. Virol., 36, 366-373.Sanger,F., Nickler,S. and Coulson,A.R. (1977) Proc. Natl. Acad. Sci. USA,

74, 5463-5467.Sanger,F., Coulson,A.R., Barrel,B.G., Smith,A.J.H. and Roe,B.A. (1980) J.

Mol. Biol., 143, 161-178.Stanley,J., Rottier,P., Davies,J.W., Zabel,P. and Van Kammen,A. (1978) Nucleic

Acids Res., 5, 4505-4522.Tabler,M. and Sanger,H.L. (1985) EMBO J., 4, 2191-2199.Tabor,S. and Richardson,C.C. (1985) Proc. Natl. Acad. Sci. USA, 82,

1074-1078.Tautz,D. and Renz,M. (1983) Anal. Biochem., 132, 14-19.Toyoda,H., Nicklin,M.J.H., Murray,M.G. and Wimmer,E. (1986) In Inouye,M.

and Sarma,M. (eds), Protein Engineering: Application in Science, Medicineand Industry. Academic Press, New York, in press.

Vos,P., Verver,J., Van Wezenbeek,P., Van Kammen,A. and Goldbach,R. (1984)EMBO J., 3, 3049-3053.

Weislander,L. (1979) Anal. Biochem., 98, 305-309.Wellink,J., Rezelman,G., Goldbach,R. and Beyreuther,K. (1986) J. Virol., 59,50-58.

Van Wezenbeek,P., Verver,J., Harmsen,J., Vos,P. and Van Kammen,A. (1983)EMBO J., 2, 941-946.

Zabel,P., Moerman,M., Lomonossoff,G., Shanks,M. and Beyreuther,K. (1984)EMBO J., 3, 1629-1634.

Zimmem,D. (1975) Nucleic Acids Res., 2, 1189-1201.

Received on November 6, 1986; revised on December 18, 1986

554